arf4  (Proteintech)

Journal: Journal of Hepatocellular Carcinoma

Article Title: Decoding ARF4 and EIF5B-Based Prognostic Signatures and Immune Landscape Following Insufficient Radiofrequency Ablation in Hepatocellular Carcinoma: Through Multi-Omics and Experimental Validation

doi: 10.2147/JHC.S517528

Figure Lengend Snippet: ( A ) SNV mutations in two genes (EIF5B and ARF4) in the risk signature model. ( B ) The co-occurrence probabilities of the two key genes and the ten most mutated genes were analyzed. ( C ) Detection of gain/loss of CNV in two key genes. ( D ) Detection of gain/loss of CNV frequency in two key genes. ( E ) Correlations between key genes and different molecular features of hepatocellular carcinoma were analyzed. ( F ) Risk genes were analyzed for correlation with immune cells, EIF5B and ARF4 were significantly and positively correlated with the majority of immune cells. ( G ) The correlation between two key genes, EIF5B and ARF4. ( H ) Feature plot of EIF5B and ARF4 expression after recognition of cell clusters by umap. ( I ) Violin plots showing the distribution of EIF5B and ARF4 in different immune cells. ( J ) The pathways in which the two key genes were mainly enriched were analyzed by GSEA.

Article Snippet: Next, tissue sections were incubated overnight at 4°C with anti-ARF4 primary antibody (A7644, Abclonal) and EIF5B (A15123, Abclonal) at a dilution of 1:200.

Techniques: Expressing

Journal: Journal of Hepatocellular Carcinoma

Article Title: Decoding ARF4 and EIF5B-Based Prognostic Signatures and Immune Landscape Following Insufficient Radiofrequency Ablation in Hepatocellular Carcinoma: Through Multi-Omics and Experimental Validation

doi: 10.2147/JHC.S517528

Figure Lengend Snippet: ( A ) Variations in mRNA expression levels of ARF4 and EIF5B in HepG2 and SNU449 cell lines, as well as mouse tissues, under control conditions and following heat treatment. ( B ) Analysis of ARF4 and EIF5B at the protein levels in HepG2 and SNU449 cell lines. ( C ) Quantification of ARF4 and EIF5B protein levels in mouse tissues before and after exposure to IRFA. ( D ) Schematic representation of the IRFA model used for constructing subcutaneous tumors in mice. ( E ) Histopathological analysis and immunohistochemical staining of ARF4 and EIF5B in HCC sections from normal tumors and tumors post-IRFA in a murine model. ( F ) Fluorescence microscopy images depicting ARF4 and EIF5B expression patterns in HepG2, SNU449 cell lines and in mouse tissues. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Next, tissue sections were incubated overnight at 4°C with anti-ARF4 primary antibody (A7644, Abclonal) and EIF5B (A15123, Abclonal) at a dilution of 1:200.

Techniques: Expressing, Control, Immunohistochemical staining, Staining, Fluorescence, Microscopy

Journal: Journal of Hepatocellular Carcinoma

Article Title: Decoding ARF4 and EIF5B-Based Prognostic Signatures and Immune Landscape Following Insufficient Radiofrequency Ablation in Hepatocellular Carcinoma: Through Multi-Omics and Experimental Validation

doi: 10.2147/JHC.S517528

Figure Lengend Snippet: Assessment of the impact of ARF4 and EIF5B knockdown on the invasive, migratory, and proliferative capacities of HepG2 and SNU449 cells. ( A and E ) RT-qPCR was employed to quantify the efficacy of ARF4 and EIF5B knockdown in HepG2 and SNU449 cells. ( B and F ) The CCK-8 assay was conducted to evaluate the proliferation of HepG2 and SNU449 cells. ( C and G ) Cell migration was assessed via transwell assays. ( D and H ) Cell invasion was assessed via transwell assays. ( I and J ) The colony-forming assay was employed to assess the proliferative potential of HepG2 and SNU449 cells. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Next, tissue sections were incubated overnight at 4°C with anti-ARF4 primary antibody (A7644, Abclonal) and EIF5B (A15123, Abclonal) at a dilution of 1:200.

Techniques: Knockdown, Quantitative RT-PCR, CCK-8 Assay, Migration

Journal: Journal of Hepatocellular Carcinoma

Article Title: Decoding ARF4 and EIF5B-Based Prognostic Signatures and Immune Landscape Following Insufficient Radiofrequency Ablation in Hepatocellular Carcinoma: Through Multi-Omics and Experimental Validation

doi: 10.2147/JHC.S517528

Figure Lengend Snippet: ( A ) Immunofluorescence staining delineated alterations in the expression profiles of EMT-related genes, especially highlighting the dynamics of E-cadherin, Snail, and vimentin (depicted in green) within HCC cells. The cellular nuclei were concurrently counterstained with DAPI (blue). ( B ) Heatmap of gene changes pre and post ablation analysis using transcriptome sequencing. ( C ) Analysis of transcriptome sequencing reveals volcanic picture in gene expression pre- and post-ablation. ( D ) Transcriptome sequencing was used to analyze the changes in the differentially expressed genes EIF5B and ARFs family. ( E and F ) GO and KEGG pathway enrichment analysis. ( G ) GSEA of ARF4 and EIF5B-associated signaling pathways. ( H ) Shared pathways between ARF4 and EIF5B. ( I ) Pathway enrichment heatmap (NES) between ARF4 and EIF5B.

Article Snippet: Next, tissue sections were incubated overnight at 4°C with anti-ARF4 primary antibody (A7644, Abclonal) and EIF5B (A15123, Abclonal) at a dilution of 1:200.

Techniques: Immunofluorescence, Staining, Expressing, Sequencing, Gene Expression, Protein-Protein interactions

Journal: Advanced Science

Article Title: Cholesterol 25‐Hydroxylase Protects Against Diabetic Kidney Disease by Regulating ADP Ribosylation Factor 4

doi: 10.1002/advs.202309642

Figure Lengend Snippet: Mass spectrometry analysis of 25‐HC interactive proteins.

Article Snippet: Empty pLKO.1 shRNA (SHC001) and human ARF4 shRNA (TRCN0000286251) plasmids were purchased from Sigma.

Techniques: Mass Spectrometry, Molecular Weight

Journal: Advanced Science

Article Title: Cholesterol 25‐Hydroxylase Protects Against Diabetic Kidney Disease by Regulating ADP Ribosylation Factor 4

doi: 10.1002/advs.202309642

Figure Lengend Snippet: 25‐HC binds to ARF4 to increase its activity. a) Western blot analysis of ARF4 after DARTS assay with different 25‐HC treatment concentrations. b) Lysates of HUVECs overexpressing CH25H were immunoprecipitated with GGA3 beads and immunoblotted for ARF4. The top panel shows the western blot immunoprecipitated protein and the bottom panel shows the total input. c) Lysates of HUVECs treated with different concentrations of 25‐HC were immunoprecipitated with GGA3 beads and immunoblotted for ARF4. The top panel shows the western blot immunoprecipitated protein and the bottom panel shows the total input. d) HUVECs were transduced with pLKO lentiviral control shRNA or shRNA against ARF4 (ARF4 shRNA). Lysates were subjected to western blot analysis using primary antibodies as indicated. e) HUVECs were transduced with pLKO lentiviral control shRNA or shRNA against ARF4 (ARF4 shRNA), as well as overexpressed with control vector or CH25H. HUVECs were transfected with Gaussia luciferase and Gaussia luciferase activity in the medium was measured. f) HUVECs were transduced with pLKO lentiviral control shRNA or shRNA against ARF4 (ARF4 shRNA), and treated with or without 25‐HC. HUVECs were transfected with Gaussia luciferase and Gaussia luciferase activity in the medium was measured.* P < 0.05, **** P < 0.0001 by 2‐way ANOVA.

Article Snippet: Empty pLKO.1 shRNA (SHC001) and human ARF4 shRNA (TRCN0000286251) plasmids were purchased from Sigma.

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Transduction, Control, shRNA, Plasmid Preparation, Transfection, Luciferase

Journal: Advanced Science

Article Title: Cholesterol 25‐Hydroxylase Protects Against Diabetic Kidney Disease by Regulating ADP Ribosylation Factor 4

doi: 10.1002/advs.202309642

Figure Lengend Snippet: ASAP1 is increased in endothelial cells of diabetic mice and negatively regulates ARF4 activity. a) ASAP1 expression in mice kidney cells by single‐cell sequencing analysis of kidneys from diabetic OVE26 mice and control mice. b) Representative ASAP1 immunofluorescence images and quantification of mean glomerular ASAP1 intensity per mouse ( n = 5 mice per group, 20–30 glomeruli scored per mouse). Scale bar = 20 µm. c) HUVECs were treated with normal glucose (NG) or high glucose (HG) and were immunoprecipitated with GGA3 beads and immunoblotted for ARF4. The top panel shows the immunoprecipitated proteins and the bottom panel shows total input. d) HUVECs were infected with Flag‐tagged ASAP1 or control vectors and then treated with either vehicle or 1 n m 25‐HC. Cells were immunoprecipitated with GGA3 beads and immunoblotted for ARF4. The top panel shows the immunoprecipitated proteins and the bottom panel shows total input. e) Measurement of Gaussia luciferase activity in the medium of HUVECs overexpressed with or without ASAP1 and treated with or without 25‐HC. f) Representative images and quantification of proximity ligation assay (PLA) between ASAP1 and ARF4 in HUVECs treated with or without 25‐HC. * P < 0.05, ** P < 0.01 by 2‐way ANOVA. **** P < 0.0001 by t ‐test. ns, non‐significant.

Article Snippet: Empty pLKO.1 shRNA (SHC001) and human ARF4 shRNA (TRCN0000286251) plasmids were purchased from Sigma.

Techniques: Activity Assay, Expressing, Sequencing, Control, Immunofluorescence, Immunoprecipitation, Infection, Luciferase, Proximity Ligation Assay

Journal: Advanced Science

Article Title: Cholesterol 25‐Hydroxylase Protects Against Diabetic Kidney Disease by Regulating ADP Ribosylation Factor 4

doi: 10.1002/advs.202309642

Figure Lengend Snippet: CH25H protects HUVECs from cell death by increasing ARF4 activity. a) HUVECs were transduced with pLKO lentiviral control shRNA or shRNA against ARF4 (ARF4 shRNA). Representative images of Giantin staining of HUVECs expressing control vector or shRNA against ARF4. Scale bar = 10 µm. b) HUVECs were transduced with pLKO lentiviral control shRNA or shRNA against ARF4 (ARF4 shRNA). Lysates were subjected to western blot analysis using primary antibodies as indicated. c) Cell viability measurement of HUVECs overexpressed with control vector or ASAP1 or/and CH25H. d) Cell viability measurement of HUVECs overexpressed with control vector or ARF4 and treated with normal glucose (NG) or high glucose (HG). ** P < 0.01, **** P < 0.0001 by 2‐way ANOVA.

Article Snippet: Empty pLKO.1 shRNA (SHC001) and human ARF4 shRNA (TRCN0000286251) plasmids were purchased from Sigma.

Techniques: Activity Assay, Transduction, Control, shRNA, Staining, Expressing, Plasmid Preparation, Western Blot

Journal: Advanced Science

Article Title: Cholesterol 25‐Hydroxylase Protects Against Diabetic Kidney Disease by Regulating ADP Ribosylation Factor 4

doi: 10.1002/advs.202309642

Figure Lengend Snippet: Protective effects of CH25H/25H in DKD. CH25H/25HC protects diabetic kidneys by maintaining ARF4 activity via inhibiting ARF4 interaction with its GAP, ASAP1. Increased ASAP1 in diabetic kidneys leads to Golgi and endothelial cell dysfunction to promote DKD.

Article Snippet: Empty pLKO.1 shRNA (SHC001) and human ARF4 shRNA (TRCN0000286251) plasmids were purchased from Sigma.

Techniques: Activity Assay

Journal: mSphere

Article Title: Toxoplasma and Plasmodium associate with host Arfs during infection

doi: 10.1128/msphere.00770-23

Figure Lengend Snippet: Internalization of host Arfs into the T. gondii PV. Representative confocal immunofluorescence images of Tg-mCh (red)-infected HeLa cells overexpressing human Arf-HA fusion proteins. Cells were stained with an anti-HA antibody (green) and nuclei were stained with Hoechst (blue). ( A ) At 32 hpi, Arf1, Arf3, Arf4, Arf5, and Arf6 were observed within the T. gondii vacuole. Yellow arrows indicate the T. gondii PV. Insets (yellow boxes) of the T. gondii PV are shown to magnify Arf puncta. ( B ) Arf1, Arf3, Arf4, Arf5, and Arf6 were visualized in uninfected and Tg-mCh -infected HeLa cells at 4, 24, and 48 hpi. The enrichment of host Arfs at the PV was observed at 4 hpi (yellow arrows) which were internalized by the end of the lytic cycle at 48 hpi. ( C ) Quantification of T. gondii PVs with at least one punctum of host protein at 4 (gray), 24 (blue), and 48 (green) hpi. Data represent mean ± SEM; n = 3 biological replicates analyzing >50 PVs per each condition. ( D ) Plot showing the mean Arf fluorescent intensity values within the PV of infected cells at 48 hpi. n = 3 biological replicates analyzing a minimum of 20 PVs per replicate. P -values display one-way ANOVA with Dunnett’s multiple comparison test for each condition compared to Arf1. ** P < 0.01; **** P < 0.0001. Scale bars are 10 µm.

Article Snippet: Primary antibodies include mouse monoclonal anti-HA (Santa Cruz Cat# sc-7392, 1:400), rabbit monoclonal anti-HA (Cell Signaling Technology Cat# 3724, 1:400), rabbit monoclonal anti-GM130 (Abcam Cat# 52649, 1:200), rabbit monoclonal anti-GBF1 (Abcam Cat# 189512, 1:300), rabbit monoclonal anti-Arf4 (Abcam Cat# AB171746, 1:100), mouse monoclonal anti-V5 (Invitrogen Cat# R96025, 1:700), goat polyclonal anti-UIS4 (Antibodies.com Cat# A121573, 1:1000), and phalloidin-iFluor 647 (abclonal Cat# ab176759).

Techniques: Immunofluorescence, Infection, Staining, Comparison

Journal: mSphere

Article Title: Toxoplasma and Plasmodium associate with host Arfs during infection

doi: 10.1128/msphere.00770-23

Figure Lengend Snippet: Arf4 and GBF1 depletion impairs P. berghei liver stage development. ( A-C ) HuH7 cells were reverse transfected (15 nM) for 48 hours with a non-targeting scramble control (siCTRL, gray) or two pooled siRNAs targeting Arf4 (blue), GBF1 (green), and Arf1 (orange). ( A ) The relative mRNA levels of HuH7 cells treated with pooled siRNAs were determined by qRT-PCR. Samples were normalized to Hs 18S and compared to cells treated with siCTRL. ( B ) Forty-eight hours post-siRNA transfection, cells were infected with Pb- Luc sporozoites. HuH7 cell viability was assessed at 48 hpi using a CellTiter-Fluor assay. Data are normalized to siCTRL. ( C ) The parasite load was assessed at 48 hpi and normalized to cells treated with siCTRL. ( D-F ) HuH7 cells were reverse transfected (15 nM) for 48 hours with a non-targeting scramble control (siCTRL, gray) or two different siRNAs targeting Arf4 (1 and 2, blue) and GBF1 (1 and 2, green). ( D ) The relative mRNA levels of HuH7 cells treated with single siRNAs were determined by qRT-PCR. Samples were normalized to Hs 18S and compared to cells treated with siCTRL. ( E ) Forty-eight hours post-siRNA transfection, cells were infected with Pb- Luc sporozoites. HuH7 cell viability was assessed at 48 hpi using a CellTiter-Fluor assay. Data are normalized to siCTRL. ( F ) The parasite load was assessed at 48 hpi and normalized to cells treated with siCTRL. ( G-I ) Cells were reverse transfected with two pooled siRNAs (15 nM) for a non-targeting scramble control, Arf4, and GBF1. The ( G ) relative infection rate at 4 hpi, ( H ) relative infection rate at 48 hpi, and ( I ) PV size at 48 hpi were assessed for a non-targeting scramble control (siCTRL, gray), Arf4 (blue), and GBF1 (green). Data for the relative infection rates were normalized to cells treated with siCTRL. ( A-I ) Data represent mean ± SEM, n = 3 biological replicates. P -values display one-way ANOVA with Dunnett’s multiple comparison test for each condition compared to siCTRL. ns = non-significant * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: Primary antibodies include mouse monoclonal anti-HA (Santa Cruz Cat# sc-7392, 1:400), rabbit monoclonal anti-HA (Cell Signaling Technology Cat# 3724, 1:400), rabbit monoclonal anti-GM130 (Abcam Cat# 52649, 1:200), rabbit monoclonal anti-GBF1 (Abcam Cat# 189512, 1:300), rabbit monoclonal anti-Arf4 (Abcam Cat# AB171746, 1:100), mouse monoclonal anti-V5 (Invitrogen Cat# R96025, 1:700), goat polyclonal anti-UIS4 (Antibodies.com Cat# A121573, 1:1000), and phalloidin-iFluor 647 (abclonal Cat# ab176759).

Techniques: Transfection, Quantitative RT-PCR, Infection, Comparison

Journal: mSphere

Article Title: Toxoplasma and Plasmodium associate with host Arfs during infection

doi: 10.1128/msphere.00770-23

Figure Lengend Snippet: Arf4 and GBF1 are associated with the P. berghei PVM. ( A ) Representative confocal immunofluorescence microscopy images of P. berghei -infected HeLa cells overexpressing Arf1-HA at 24 and 48 hpi. Arf1-HA did not colocalize with the P. berghei PVM resident protein UIS4. Cells were stained with anti-HA (red), anti-USI4 (green), and nuclei were stained with Hoechst (blue). ( B and C ) P. berghei -infected HuH7 cells at 24 and 48 hpi were stained with ( B ) anti-Arf4 (red) or ( C ) anti-GBF1 (red). Cells were stained with anti-UIS4 (green) and nuclei were stained with Hoechst (blue). Scale bars are 10 µm. ( D and E ) The percentage of P. berghei vacuoles with Arf4 or GBF1 protein accumulation was assessed using Z-stacks of P. berghei -infected HuH7 cells at 48 hpi with Imaris software. ( D ) Protein accumulation to UIS4 was scored positive if the amount of Arf4 or GBF1 spots at the vacuole was statistically greater than a random distribution of protein within the host cell. ( E ) The attraction distance was calculated for vacuoles with a positive accumulation of Arf4 or GBF1 to the UIS4 surface. The value represents the distance from the UIS4 surface where there was a statistically greater accumulation of Arf4 or GBF1 than a random simulation. Data represent mean ± SEM, n = 3 biological replicates analyzing 20 PVs for each condition.

Article Snippet: Primary antibodies include mouse monoclonal anti-HA (Santa Cruz Cat# sc-7392, 1:400), rabbit monoclonal anti-HA (Cell Signaling Technology Cat# 3724, 1:400), rabbit monoclonal anti-GM130 (Abcam Cat# 52649, 1:200), rabbit monoclonal anti-GBF1 (Abcam Cat# 189512, 1:300), rabbit monoclonal anti-Arf4 (Abcam Cat# AB171746, 1:100), mouse monoclonal anti-V5 (Invitrogen Cat# R96025, 1:700), goat polyclonal anti-UIS4 (Antibodies.com Cat# A121573, 1:1000), and phalloidin-iFluor 647 (abclonal Cat# ab176759).

Techniques: Immunofluorescence, Microscopy, Infection, Staining, Software

Journal: Cell death & disease

Article Title: GOLPH3 promotes endotoxemia-induced liver and kidney injury through Golgi stress-mediated apoptosis and inflammatory response.

doi: 10.1038/s41419-023-05975-x

Figure Lengend Snippet: Fig. 2 LPS-induced endotoxemia promotes Golgi stress and inflammatory response in acute liver and kidney injury. A Immunofluorescence staining of GM130 (green), a marker of Golgi morphology, in the liver tissues was assessed by confocal microscopy and quantification of cells with fragmented Golgi from each group is shown (n = 3). Scale bar, 20 µm. B Golgi functional and structural proteins (GRASP65, ARF4, P14KIIIβ, and MYO18A) were examined using western blot analysis in liver tissues and relative expression was shown over β-actin as loading control (n = 3–5). C Relative mRNA levels of Grasp65, Arf4, Creb3, and Myo18a were determined by real-time PCR analysis in liver tissues, and relative mRNA expression was normalized to that of GAPDH (n = 3–5). D Relative mRNA levels of pro-inflammatory cytokines (Tnfα, IL-1β, and IL-6) were determined by real-time PCR analysis in liver tissues and relative mRNA expression was normalized to that of GAPDH (n = 4). E–H As described above, the experiments in (A), (B), (C), (D) were similarly performed in kidney tissues. The data are presented as mean ± SEM. Two-tailed Student’s t-test was used, *p < 0.05 versus Control.

Article Snippet: The membranes were incubated with primary antibodies against GOLPH3, Golgi reassembly-stacking protein 1 (GRASP65) and ADP-ribosylation factor 4 (ARF4) (Proteintech), phosphatidylinositol 4-kinase III beta (PI4KIIIβ) (Abcam, Cambridge, UK), MYO18A (Santa Cruz Biotechnology, Dallas, TX, USA), phosphorylated nuclear factor kappa B (p-NF-κB) p65, NF-κB p65, phosphorylated NF-κB inhibitory subunit alpha (p-IκBα), IκBα, p-AKT, AKT, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) (Cell Signaling Technology, Danvers, MA, USA) and β-actin (Sigma-Aldrich) in the blocking solution at 4 °C overnight.

Techniques: Staining, Marker, Confocal Microscopy, Functional Assay, Western Blot, Expressing, Control, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Cell death & disease

Article Title: GOLPH3 promotes endotoxemia-induced liver and kidney injury through Golgi stress-mediated apoptosis and inflammatory response.

doi: 10.1038/s41419-023-05975-x

Figure Lengend Snippet: Fig. 2 LPS-induced endotoxemia promotes Golgi stress and inflammatory response in acute liver and kidney injury. A Immunofluorescence staining of GM130 (green), a marker of Golgi morphology, in the liver tissues was assessed by confocal microscopy and quantification of cells with fragmented Golgi from each group is shown (n = 3). Scale bar, 20 µm. B Golgi functional and structural proteins (GRASP65, ARF4, P14KIIIβ, and MYO18A) were examined using western blot analysis in liver tissues and relative expression was shown over β-actin as loading control (n = 3–5). C Relative mRNA levels of Grasp65, Arf4, Creb3, and Myo18a were determined by real-time PCR analysis in liver tissues, and relative mRNA expression was normalized to that of GAPDH (n = 3–5). D Relative mRNA levels of pro-inflammatory cytokines (Tnfα, IL-1β, and IL-6) were determined by real-time PCR analysis in liver tissues and relative mRNA expression was normalized to that of GAPDH (n = 4). E–H As described above, the experiments in (A), (B), (C), (D) were similarly performed in kidney tissues. The data are presented as mean ± SEM. Two-tailed Student’s t-test was used, *p < 0.05 versus Control.

Article Snippet: The membranes were incubated with primary antibodies against GOLPH3, Golgi reassembly-stacking protein 1 (GRASP65) and ADP-ribosylation factor 4 (ARF4) (Proteintech), phosphatidylinositol 4-kinase III beta (PI4KIIIβ) (Abcam, Cambridge, UK), MYO18A (Santa Cruz Biotechnology, Dallas, TX, USA), phosphorylated nuclear factor kappa B (p-NF-κB) p65, NF-κB p65, phosphorylated NF-κB inhibitory subunit alpha (p-IκBα), IκBα, p-AKT, AKT, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) (Cell Signaling Technology, Danvers, MA, USA) and β-actin (Sigma-Aldrich) in the blocking solution at 4 °C overnight.

Techniques: Staining, Marker, Confocal Microscopy, Functional Assay, Western Blot, Expressing, Control, Real-time Polymerase Chain Reaction, Two Tailed Test